ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 4, с. 391-395
UDC 577.154
INDUCING EFFECT OF SALICIN FOR EXTRACELLULAR ENDOGLUCANASE SYNTHESIS IN Rhizopus oryzae PR7 MTCC 9642 © 2013 M. Karmakar and R. R. Ray
Microbiology Research Laboratory, Department of Zoology, Molecular Biology & Genetics, Presidency University, Kolkata, I700 073, India e-mail: rina_ray64@yahoo.co.in Received September 20, 2012
The induction of endoglucanase of Rhizopus oryzae PR7 MTCC 9642 has been observed in cultivation medium using a batch-fermentation technique. The effect of various experimental parameters such as use various carbon sources as inducers, concentration of inducer and effect of different additives were investigated. Sali-cin at concentrations from 0.25 to 0.75% (w/v) was found to bring about a remarkable increase in endoglucanase synthesis when used as a sole carbon source and thought to act as a gratuitous inducer of the enzyme. Endoglucanase activity increased within 24 h after the addition of salicin, reached maximum after 48 h and maintained high level even after 120 h of fungal growth. Repression of enzyme synthesis by glucose could partially be restored by addition of salicin at an early phase of growth. The carboxymethyl cellulose induced enzyme was inhibited by cyclohexamide and ethidium bromide and partially recovered by the salicin which indicated that salicin might act at the transcriptional but not translational level.
DOI: 10.7868/S0555109913040077
Cellulose is the most abundant compound that contains only a-1,4-linkages [1]. An important feature of cellulose, relatively unusual in the polysaccharide world, is its crystalline structure [2] and the insoluble, recalcitrant nature of cellulose represents a challenge for cellulase systems. Cellulase enzymes, which can hydrolyze cellulose forming glucose and other cel-lo-oligosaccharides, can be divided into 3 types: endoglucanase (endo-1,4-a-D-glucanase, EC 3.2.1.4); cellobiohydrolase or exoglucanase (exo-1,4-a-D-glu-canase, EC 3.2.1.91) and P-glucosidase (1,4-P-D-glucosidase, EC 3.2.1.21) [3]. Cellulase systems are not merely an agglomeration of enzymes representing these enzyme groups but rather act in a coordinated manner to efficiently hydrolyze cellulose [2].
Microorganisms have adapted different approaches to effectively hydrolyze cellulose, naturally occurring in insoluble particles or imbedded within hemicellulose and lignin polymers [4]. Cellulases from aerobic fungi have received more study than any other physiological group, and fungal cellulases currently dominate the industrial applications ofcellulases [5—7]. Cellulases are induced in most of fungi only when cellulose or an inducer exists [8]. Cellulolytic filamentous fungi (and actinomycete bacteria) have the ability to penetrate cellulosic substrates through hyphal extensions, thus often presenting their cellulase systems in confined cavities within cellulosic particles [9].
Cellulolytic capability is well represented among the various subdivisions of aerobic fungi of which As-comycetes, Basidiomycetes, and Deuteromycetes
contain large numbers of cellulolytic species and the genus Mucor of Zygomycetes was found to possess significant activity of this enzyme [2], however very little is known about the induction of cellulase system in Rhizopus.
Since the natural inducer cellulose is insoluble, several studies were performed to determine how an insoluble polymer, which cannot transfer into the cell, would initiate cellulase production [10]. It was postulated [11—13] that low basal level of cellulases is formed, which can start the degradation of cellulose and thereby release small amounts of oligosaccha-rides, which can induce further cellulase biosynthesis. After the degradation of cellulose, the liberated end product causes catabolite repression which prevents the fungus from synthesizing an excess amount of cellulose [2] through a negative regulation at the transcriptional level. Although a number of inducers were reported for fungal cellulase synthesis, effect of non cellulosic substance in uninduced or in catabolically repressed fungus was not investigated. The aim of the study was the induction and catabolite repression of endoglucanase synthesis in Rhizopus oryzae highlighting the role of salicin as inducer with its plausible mechanism of action.
MATERIALS AND METHODS
Microorganism. Rhizopus oryzae PR7 MTCC 9642, an endoglucanase producing strain was isolated from the decaying vegetation enriched soil of India.
U/mL
h
Fig. 1. Production of the R. oryzae PR7 MTCC 9642 en-doglucanase in presence of various carbon sources: CMC (1), cellobiose (2), glucose (3), salicin (4), and CMC+sali-cin (5) in the cultivation medium.
The strain was identified by Institute of Microbial Technology (IMTECH), Chandigarh, India and deposited in Microbial Type Culture Collection as Rhizopus oryzae MTCC 9642.
Chemicals. All chemicals used were of analytical grade.
Cultivation of the fungus. The strain R. oryzae PR7 MTCC 9642 was cultivated in 100 mL Erlenmeyer flasks each containing 20 mL of basal medium (BM) composed of (g/L): peptone - 0.9; (NH4)2HPO4 -0.1; KCl - 0.1; MgSO4 • H2O - 0.1 and carboxymethyl cellulose (CMC) - 0.5 (pH 6.0).
Preparation of inocula. The fungus was grown in 1% PDA plates, for 48 h at 28-30°C. The inocula were prepared by making hyphal discs (0.5 cm diameter). Each disc was used to inoculate 20 mL of medium [14].
Effect of various factors. The culture flasks (100 mL) with 20 mL medium were cultivated at various temperatures (4°C-40°C) for 0-144 h. To determine the effect of carbon sources, CMC was replaced by various polysaccharides and simple sugars. BM was supplemented with various additives (at concentration 10 mM). Antifungal agents (0.25 ^g/mL) were added in the culture flask to test the susceptibility of the fungus.
Enzyme assay. The culture broth of filamentous form of fungus was filtered through filter paper (Whatman No. 1, USA) and filtrate was used as crude enzyme whereas the culture broth of the yeast form was centrifuged at 10000 rpm for 5 min and the supernatant was used as the crude enzyme. To measure the activity of endoglucanase, the assay mixture (1 mL) containing an equal volume of properly diluted enzyme and 1.0% (w/v) CMC in 50 mM phosphate buffer
(pH 7.0) was incubated at 37°C for 10 min. The reducing sugar released was measured by the dinitrosalicylic acid method [15] taking glucose as standard. Blanks were prepared with inactivated enzymes treated by exposing at 60°C for 15 min. One unit of endoglucanase was defined as that amount of enzyme that liberated 1 ^mole of glucose per mL per min of reaction.
Protein estimation. The extracellular protein was estimated by the Lowry method. The supernatant of yeast form was scanned spectrophotometrically at 250-300 nm range.
Each experiment was performed in triplicate and their values were averaged.
RESULTS AND DISCUSSION
R. oryzae was found to produce all components of enzyme complex hydrolyzing cellulose, namely endo-glucanase [16], exoglucanase [17] and P-glucosidase [18], of which endoglucanase was the most pronounced. The regulation of endoglucanase synthesis by induction and catabolite repression in R. oryzae PR7 MTCC 9642 studied in batch fermentations indicated that a basal level of endoglucanase was synthesized by the fungus even in absence of any carbohydrate source. The observation was similar to that of Hulme and Stranks [19], who working with several cellulolytic fungi, came to the conclusion that cellu-lase is not formed as a response to any inducing substance. According to Carle-Urioste with coworkers [13] and Suto and Tomita [8], a basal level of constitutive cellulase might degrade cellulose into cello-oli-gosaccharides, and they trigger the induction after entry into the cell.
It has been proposed that a low constitutive level of cellulase expression in R. oryzae PR7 MTCC 9642 is responsible for the formation of an inducer from cellulose. In this fungus, low constitutive level of endoglucanase expression could be induced by CMC followed by gentibiose, similar to that reported earlier for microorganisms like Hypocrea jecorina, Penicillium purpurogenum and Chaetomium erraticum [8, 20]. It was revealed that salicin caused about a 3.5 times increase in this level.
On the other hand, although CMC acted as the inducer, it was thought that it could not trigger the induction directly because of its insolubility [8] and could not lead to that degree of endoglucanase synthesis like that of salicin (Fig. 1). The level of induction was much higher in the salicin-supplemented culture medium than that of the medium supplemented with salicin along with CMC, which indicated CMC or its derivative must have some repressive effect that actually controlled the enzyme synthesis.
The concentration of salicin played an important role in inducing the R. oryzae endoglucanase. The best concentration of salicin was found to be 0.75% (w/v), after which it gradually declined (Fig. 2) indicating
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that above a certain saturation level it failed to operate the induction mechanism of the enzyme.
The maximum induction of endoglucanase of R. oryzae PR7 MTCC 9642 in presence of different types of carbon sources was revealed after 48 h of cultivation, although salicin could brought about an abrupt increase in enzyme production (Fig. 1). Salicin (C13H18O7) being an alcoholic P-glucoside containing D-glucose could maintain the enzyme synthesis at a high level even after 120 h of growth. This fact indicated that it must have acted as a non-metabolizable trigger that could not be degraded and hydrolyzed by the cellulase system of the fungus.
Cultivation of the fungal cell in presence of only glucose or cellobiose, failed to enhance the endoglu-canase synthesis from (Fig. 1), but these compounds could not be regarded as the end product inhibitors, rather as c
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